Comprehensive Transcriptome Analyses Reveal Candidate Genes for Variation in Seed Size/Weight During Peanut (Arachis hypogaea L.) Domestication

Abstract

Seed size/weight, a key domestication trait, is also an important selection target during peanut breeding. However, the mechanisms that regulate peanut seed development are unknown. We re-sequenced 12 RNA samples from developing seeds of two cultivated peanut accessions (Lines 8106 and 8107) and wild Arachis monticola at 15, 30, 45, and 60 days past flowering (DPF). Transcriptome analyses showed that ∼36,000 gene loci were expressed in each of the 12 RNA samples, with nearly half exhibiting moderate (2 ≤ FPKM < 10) expression levels. Of these genes, 12.2% (4,523) were specifically expressed during seed development, mainly at 15 DPF. Also, ∼12,000 genes showed significant differential expression at 30, 45, and/or 60 DPF within each of the three peanut accessions, accounting for 31.8-34.1% of the total expressed genes. Using a method that combined comprehensive transcriptome analysis and previously mapped QTLs, we identified several candidate genes that encode transcription factor TGA7, topless-related protein 2, IAA-amino acid hydrolase ILR1-like 5, and putative pentatricopeptide repeat-containing (PPR) protein. Based on sequence variations identified in these genes, SNP markers were developed and used to genotype both 30 peanut landraces and a genetic segregated population, implying that EVM0025654 encoding a PPR protein may be associated with the increased seed size/weight of the cultivated accessions in comparison with the allotetraploid wild peanut. Our results provide additional knowledge for the identification and functional research into candidate genes responsible for the seed size/weight phenotype in peanut.

Keywords: SNP marker; peanut (Arachis hypogaea L.); pentatricopeptide repeat protein (PPR); seed size/weight; transcriptome analysis.

FIGURE 1

Seed phenotypes of three peanut accessions at four seed developmental stages. (A) Seed samples at 15, 30, 45, and 60 DPF from A. monticola (A. mon), Line 8106, and Line 8107, scale bar = 1 cm. Panels (B,C) show the changes in average size and weight for seeds from A. mon, Line 8106, and Line 8107 from 15 DPF to maturity. One-way analysis of variance was performed. Significant level, α = 0.05.

FIGURE 2

Expressed genes in the three peanut accessions at four seed developmental stages. (A) Proportion of genes expressed at five different levels (based on FPKM) during the four seed developmental stages (15, 30, 45, and 60 DPF) for A. monticola, Line 8106, and Line 8107. Panels (B–D) are Venn diagrams showing the number of genes with shared or stage-specific expression at the four seed developmental stages (15, 30, 45, and 60 DPF) in A. monticola, Line 8107, and Line 8106, respectively.

FIGURE 3

Differential gene expression at the four seed developmental stages in each of the three peanut accessions. (A) The number of up- and down-regulated genes shared among three seed developmental stages (30, 45, and 60 DPF) for A. monticola, Line 8106, and Line 8107. (B) Heatmap showing the relative expression of plant hormone signaling pathway-related DEGs. (C) Expression levels of six representative genes from 15 to 60 DPF that are enriched in the plant hormone signaling pathway.

FIGURE 4

DEGs between A. monticola and two cultivated peanut accessions at four seed developmental stages. Up-/down-regulated genes were detected in Line 8107 (A) and Line 8106 (B) at 15, 30, 45, and 60 DPF in pairwise comparisons with the wild peanut species A. monticola. Some GO terms were significantly enriched in the DEGs from Lines 8107 and 8106, and are shown in panels (C,D), respectively. T19–T22, and T23–T26 indicate the seed samples at 15, 30, 45, and 60 DPF from Line 8106 and Line 8107, respectively.

FIGURE 5

Expression levels of identified candidate genes associated with peanut seed size/weight in A. monticola (A. mon), Line 8106, and Line 8107. The genes EVM0023227, EVM0062133, and EVM0031048 encode topless-related protein 2, transcription factor TGA7, and IAA-amino acid hydrolase ILR1-like 5, respectively. The other genes encode pentatricopeptide repeat-containing proteins.

FIGURE 6

EVM0025654 is associated with increased seed size/weight in cultivated peanut. (A) 100-seed weights for A. monticola and three cultivated peanut accessions. (B) The marker SNP564 was used to genotype these cultivated peanut lines and the SNP locus at position 490 is shown in blue. (C) Seeds of recombinant inbred line (RIL) population (F₆) derived from a cross of Zp06 (100-seed weight, 106.2 g) and A. monticola with a 100-seed weight of 19.4 g. Large, some RILs have large seeds; small, others lines have small seeds. (D) Box plot for seed weight of the RILs having G/G, G/C, and C/C genotype at the SNP locus (111201604 bp) in EVM0025654. A Student’s t-test indicated a significant difference (n = 5 plants) in panel (A). ^∗∗P < 0.01. All data are given as mean ± SD. Bar = 10 mm.